Glycopeptide extract

ABSTRACT

Immunostimulating glycopeptides obtainable from the cell walls of immunostimulating bacteria belonging to the Mycobacteriaceae or Actinomycetaceae Family and methods of isolating the glycopeptides.

This application is a continuation-in-part of U.S. patent applicationSer. No. 445,309 filed Feb. 22, 1974, now U.S. Pat. No. 4,010,257.

The present invention relates to glycopeptides having pharmacologicalactivity, the extraction of the glycopeptides from bacterial cells andtheir adaptation for medicinal use.

It has previously been known that certain bacteria belonging to theActinomycetaceae or Mycobacteriaceae Families of Prevot's classification(1), have immunostimulating properties.

As used herein `immunostimulating` means capable of stimulating anon-specific immune response in mammals, and birds that is, capable ofconferring on mammals and birds a resistance to tumours and variouspathogens, such as viruses, bacteria (12), and parasites (13), in theabsence of antigenic affinity between the pathogen and the products ofimmunostimulation; as well as behaving as an adjuvant in mammals andbirds.

It has now been found that glycopeptides having immunostimulatingproperties may be obtained from the abovementioned bacteria.

The present invention provides an immunostimulating glycopeptidesuitable for use in conferring on mammals or birds a resistance totumours and various pathogens which glycopeptide is obtainable from thecell wall of an immunostimulating bacterium belonging to theActinomycetaceae or Mycobacteriaceae families of Prevot'sclassification, which glycopeptide is substantially insoluble in water,physiological saline, methanol, ethanol, phenol, chloroform, dioxan,pyridine, dimethylformamide, diethylene glycol, and hexane; has anabsorption in the Infra-red spectrum at 1550 and 1660 cm⁻¹ consistentwith the presence of peptide; has an absorption in the Infra-redspectrum between 950 and 1100 cm⁻¹ with a maximum absorption at 1050 to1070 cm⁻¹ consistent with the presence of carbohydrate; has little or nolipid absorption in the Infra-red spectrum at 2850 to 2950 cm⁻¹ ; andwhich glycopeptide, after intravenous administration, increases theliver and spleen weight of mice and increases the life expectancy ofmice with tumours.

Genera belonging to one or other of the Actinomycetaceae orMycobacteriaceae families tht may be mentioned are Actinomyces,Nocardia, Streptomyces, Micromonospora, Corynebacterium (also known asPropionibacterium) both aerobic and anaerobic species, Actinobacterium(also known as Bifidobacterium), Erysipelothrix, Listeria andMycobacterium.

Species belonging to the Genus Corynebacterium which may be mentionedinclude C. anaerobium, C. granulosum, C. liquefaciens, C. pyogenes, C.lymphophilum, C. hepatodystrophicans, C. adamsoni, C. parvum, C. avidum,C. diphtheroides, C. renale cuniculi, C. acnes, C. equi, and C. ovis.

There is considerable controversy concerning the taxonomy of theCorynebacteria. Nevertheless any references to the species C. parvumherein are intended to include the strain having Wellcome CultureCollection number CN 6134 which strain was obtained from the PasteurInstitute and has the Pasteur Institute's Culture number 4685, and haspreviously been described as C. anaerobium (see for example, Ref. 9).

The composition of the glycopeptides of the present invention is subjectto variation due to such factors as genetic variation depending on theparticular strain used as a source of bacterial cells and the particularmethods used in the culture of those cells. Variation may also occurarising out of the degree of purity achieved by the isolation processused, for example, due to incomplete removal of external proteinattached to the glycopeptides.

Desirably glycopeptides of the present invention comprise from 30 to45%, preferably about 40%, by weight of carbohydrate.

Preferably the glycopeptides comprise according to micronalysis, from 38to 43% Carbon, from 7 to 10% Nitrogen and from 5 to 7% of Hydrogen, thepercentages being by weight of the dry glycopeptide.

The glycopeptides of the present invention may be extracted from thebacterial cells by preparing a culture of the bacterial cells;recovering cultured bacterial cells from the culture; optionally lysingthe cultured bacterial cells to produce a lysate thereof; and subjectingthe cultured bacterial cells or lysate thereof to biphasic solventextraction so as to yield the glycopeptide as insoluble residue.

The bacterial cells from which the glycopeptides of the presentinvention are obtained, may be obtained from any known source. Ingeneral the bacterial cells may be initially obtained from mammals orbirds infected with the required bacterium, for example, C. parvum maybe obtained from the blood of patients suffering from a septicaemiawhilst C. renale cuniculi is often associated with normal mammaliankidneys. Some bacteria, however can be obtained from more than onehabitat, for example, C. parvum may also be obtained from the skin ofsome normal humans.

Bacterial cells which have immunostimulating properties (from which theglycopeptides of the present invention are obtainable) may be readilyidentified by their ability to increase spleen and liver weight in miceand/or their adjuvant activity in mice (9) and their ability to produceresistance to tumours in mice (14).

Any culture of the bacterial cells prepared by methods already known,may be used as a material for the extraction of the glycopeptide.Advantageously freshly isolated strains are stored in a freeze-driedcondition before being cultured in a nutrient medium, such asRobertson's cooked meat medium (3) supplemented with 1% w/v glucose and5% v/v horse serum. The culture is desirably maintained at a temperatureabove ambient, for example, about 37° C for several days, conveniently 5to 7 days and preferably without disturbance of the culture medium. Inorder to increase the yield of culture, the products of the initialculture may be used to seed further quantities of growth medium, forexample, a medium containing 1% w/v glucose, wherein growth takes placeunder similar conditions. Other suitable growth media includeTodd-Hewitt broth (4) and Thioglycollate broth (5). In order that themaximum yield of cells is obtained in the minimum time, successivesubculture of the cells is required and each subculture is preferablyeffected in the logarithmic phase of growth and on each occasion theinoculum is preferably about 10% by volume of the succeeding culturethough for the last stage the inoculum is conveniently 2% to 3% byvolume of the succeeding culture.

The cultured bacterial cells may be recovered from the culture medium byany conventional process for harvesting such media, for example, bycentrifugation. Desirably the cultured bacterial cells are thenrepeatedly washed, for example with aqueous 0.85% w/v sodium chloride orwith distilled water, prior to extraction.

Lysis of the cultured bacterial cells may be effected by anyconventional technique for the lysis of cells, for example, by the useof physico-chemically acting agents such as hypertonic buffers withmechanical agitation of the viscous lysate; or mechanical lysis, forexample by use of ultrasonic sound waves; by intracellular cavitation;by passage at low temperature and high pressure through a narrow orificeas in a press such as the Hughes press (6); or by mechanical agitationin a homogeniser or grinder (7). Desirably the broken cell walls areseparated from the other constituents of the lysate, for example, bycentrifugation. Centrifugation may be effected at 15,000 to 25,000, forexample, 20,000g for about 30 to 90 minutes, for example, 1 hour.Conveniently a preliminary centrifugation is carried out at a low speed,for example at about 300g. for 3 minutes, the supernatant from thisstage then being used for the main centrifugation stage at higher speed,intact cells remaining in the sediment.

Biphasic solvent extraction of the broken cell walls, and/or culturedwhole bacterial cells, and/or the lysate thereof so as to yield theglycopeptide is effected with aqueous aryl alcohol and the glycopeptideisolated from the cell constituents not extracted in the aqueous phase.As used herein `aryl alcohol` means a compound wherein a hydroxy nucleusis directly attached to an aromatic nucleus, for example, an optionallysubstituted phenol such as a cresol, but most preferably unsubstitutedphenol.(see Example, Ref.16)

The biphasic solvent extraction is effected by contacting with water andthe aryl alcohol for a period of time at a temperature of from 0° C to100° C, preferably from 15° to 70° C, conveniently with stirring. Asuitable period of time is from a few minutes to a few hours dependingon the temperature employed. In the case where phenol is used, aparticularly suitable temperature is 68° C at which stirring may becarried out for 15 minutes.

The concentration of aryl alcohol in the aryl alcohol-water mixture maybe varied somewhat, it being necessary to bear in mind that one mustultimately obtain a 2-phase system, each phase being fully saturatedwith the other. In the case of phenol, the phenol concentration in thephenol-water mixture may be from 10 to 90% v/v, but most preferably from40 to 55% v/v.

The mixture is then centrifuged at about 5,000 to 10,000g for 45 to 15minutes, for example, at 8,000g for 30 minutes, to yield four layers: asolid pellet containing cell debris, above which are in turn thealcoholic phase, a white interphase region, and the aqueous phase. Inthe case of a process not involving lysis, little or no material ispresent in the interphase region, centrifugation yeilding only threelayers: a solid pellet containing unbroken cells, above which are inturn the alcoholic phase, and the aqueous phase.

The aqueous phase and alcoholic phases are discarded and desirably thebiphasic solvent extraction is repeated on the remaining materialpreferably until the aqueous and alcoholic phases, after centrifugation,are colourless (or in the case of a coloured alcohol, are unchanged incolour in the course of extraction). Alternatively only the aqueousphase is discarded, the remaining material including the alcoholicphase, being reextracted if desired.

The material remaining after the aqueous phase (and if desired, thealcoholic phase) has been discarded is then desirably dialysed againstwater to remove the aryl alcohol (or residual aryl alcohol,respectively). Dialysis is effected for several days, desirably at atemperatue below ambient, for example, at about 4° C.

After dialysis a sediment is obtained comprising the following layers: adark coloured lower layer (not present when the alcoholic phase has beendiscarded after centrifugation), a brown cream coloured layer having anoily consistency and an upper layer comprising a white finely dividedsuspension which may then be collected to yield the immunostimulatingglycopeptides of the present invention which are desirably washed withdistilled water or physiological saline (0.85% w/v aqueous sodiumchloride).

The glycopeptides of the present invention may be used therapeuticallyor prophylactically in conferring on mammals and birds a resistance totumours and various pathogens, inhibiting the growth of such tumours andpathogens and/or decreasing their extent, by administration of aneffective therapeutic or prophylactic dosage of the glycopeptides.Tumours that may be mentioned in this context include leukaemias,Hodgkin's disease, carcinoma of the lung, bladder and breast, melanomaand Burkitt's lymphoma. Bacterial pathogens that may be mentionedinclude Bordetella pertussis and Brucella abortus. Parasitic pathogensinclude protozoal pathogens such as malaria.

The magnitude of the therapeutic or prophylactic dosage of glycopeptidewill of course vary with the nature and severity of the tumour of thepathogenic infection concerned, with the particular glycopeptide and itsroute of administration. In general the dosage range lies from 0.0001 to20, preferably 0.1 or 0.5 to 10, and most preferably from 0.7 to 2mg ofglycopeptide per kg. bodyweight of the mammal or bird to which theglycopeptide is to be administered.

Any conventional mode of administration may be used at the discretion ofthe attending physician and depending on the nature and location of thetumour or pathogenic infection concerned. Thus administration may be bythe oral or pulmonary route though usually administration is byinjection subcutaneously, intramuscularly, intraperitoneally or,preferably, intravenously. If desired "injection" may comprise infusionover an extended period of time, for example, over several hours.

The glycopeptides may be formulated into a pharmaceutical compositionfor use in a method of treatment or prophylaxis as describedhereinbefore, by any conventional pharmaceutical technique involvinginter alia the step of admixture of the glycopeptides with one or morepharmaceutically acceptable carriers. The nature of the composition willof course depend on the route chosen for administration. Thus for oraladministration, a composition of a glycopeptide of the present inventionmay be presented as a discrete unit such as a capsule, cachet or tableteach containing a predetermined amount of the glycopeptide; as a powderor granules; or as a suspension in an aqueous liquid, a non-aqueousliquid an oil-in-water emulsion or a water-in-oil emulsion. Forpulmonary administration, a composition of a glycopeptide of the presentinvention may be present as an inhalation composition.

A pharmaceutical composition of the present invention intended foradministration by injection comprises a glycopeptide of the presentinvention suspended in an inert pharmaceutically acceptable liquiddiluent. The compositions for injection must of course be sterile andisotonic with the blood of the mammal or bird into which they are to beadministered. A suitable diluent is water and a suitable injectioncomposition for use in humans is a suspension of the glycopetide inphysiological siline (0.85% w/v aqueous sodium chloride). Advantageouslythe glycopeptide is suspended in a sterile diluent under asepticconditions. Sterilisation of the injection compositions may be effectedby conventional techniques, for example, by the addition of formalin toa concentration of about 0.5% v/v. Desirably a preservative, forexample, thiomersalate conveniently at a concentration of 0.01% w/v isincluded in the injection compositions.

The injection compositions of the present invention may be renderedisotonic by any conventional technique, for example, by dialysis againsta salt solution that is isotonic with the blood of the mammal or bird tobe injected. Any salt solution suitable for use as an injection medium,for example, physiological saline (0.85% w/v aqueous sodium chloride) orisotonic phosphate buffer may be used.

The injection compositions may be prepared either as a concentratedinjection composition for dilution prior to use or as an injectioncomposition ready for use. In the latter case the concentration of theimmunostimulating glycopeptide is desirably from 20 to 2mg/ml;preferably of the order of 7mg/ml. Conveniently the injectioncompositions may be freeze-dried, for example, from 5% w/v aqueoussucrose, for reconstitution with water just before use.

A further use of glycopeptides of the present invention is as anadjuvant; or as an ingredient in a vaccine having another activeingredient. An example of such a use, is the substitution of theglycopeptide for the heat killed bacterial element of Freund's completeAdjuvant (a water-in-oil emulsion of an active ingredient in a mineraloil also containing heat killed bacteria), and an example of a vaccinecontaining the glycopeptide as an adjuvant is a vaccine for immunisationagainst Brucella.

In order that the present invention may be more fully understood, thefollowing examples are given, purely by way of illustration and are notto be construed as limiting the scope of the present invention.

EXAMPLE 1 Isolation of Immunostimulating glycopeptide of C. parvum cells

A. preparation of cultured C. parvum cells

Freeze dried cells of C. parvum (0.2 to 0.5mg) (Wellcome Culture No. CN6134) were seeded into a screwed cap bottle containing Robertson'scooked meat medium (20ml) (3) supplemented with glucose (1% w/v) andhorse serum (5% v/v). After static incubation of 37+ C for 6 days,incubation was continued for 6 days in 2 liter capacity bottles (1800mlof broth) and then for about 7 days in 15 liter capacity bottles. Ineach case the volume of seed mixture used about 10% by volume of thebroth to be seeded, except for the last stage when a 21/2% inoculum wasused.

The cultured cells were then harvested by subjecting the final culturemixture to centrifugation at 2000g, for 2 hrs. The supernatant wasdiscarded and the cells washed repeatedly by resuspending the sedimentedin cells in physiological saline and centrifuging the suspension asbefore.

B. cell lysis

The washed cells (10g wet weight) were mixed with Ballotini glass beads(35g of No.14) and a small quantity of physiological saline about 1ml).

The mixture was then subjected to mechanical agitation in a Novotnydisintegrator (8) for 15 minutes with the paddle rotating at 2000 r.p.m.The resulting slurry was then diluted with a further quantity ofphysiological saline (ca. 10ml) and filtered in a coarse sintered glassseparator to remove the glass Ballotini. The residue was resuspended inphysiological saline (ca. 10ml) and centrifuged at 20,000g for 1 hour tosediment out the broken cell walls.

C. extraction of C. parvum cell walls

To the C. parvum broken cell walls (20g wet weight) were added distilledwater (350ml) and aqueous phenol (0% w/v, 350ml), both at 68° C. Themixture was incubated at 68° C for 15 minutes constant stirring, cooledin an ice bath and the mixture then centrifuged at 8000 g for 30 mins at4° C, Four distinct layers were observed in the centrifuge vessel, apellet, a lower phenolic phase, a white interphase layer, and an upperaqueous phase. The aqueous phase was discarded and the remaining layersresuspended in a further quantity of distilled water (350ml) at 68° Cand re-extracted as before. Again the upper aqueous phase was discardedand the remaining layers were then combined and dialysed against runningtap water for 4 days followed by distilled water at 4° C for a further 3days in order to remove the phenol.

After 1 week a sediment of three layers was present in the dialysistubing. A lower dark coloured layer comprised broken cells bound up withDNA. A second browncream coloured layer had an oily constitency (both ofthese layers had little or no immunostimulating activity). The upperwhite layer was collected, after water had been removed from thedialysis tubing, to give the glycopeptide, (hereinafter referred to asglycopeptide A) of which the properties are described herein below.

Physical Properties

The glycopeptide possessed the following properties:

a. It was slightly soluble in dimethyl sulphoxide. It was insoluble inwater, phenol, ethanol, methanol, chloroform, dioxan, pyridine,dimethylformamide, diethylene glycol, hexane, acetone, ether andphysiological saline;

b. It was substantially lipid-free (J. Falch et al J. Biol. Chem. 226497 (1957));

c. When suspended in a phenol-water mixture, it became concentrated inthe interphase region;

d. It had a chemical composition as determined by microanalysis, theamounts representing percentages by weight: Carbon 39.03; Hydrogen 6.24;Nitrogen 9.03; Phosphorus Trace; Ash 5.4; Silica 1.48; Sulphur below0.2; and Oxygen, by difference, of the order of 40. Carbon, Hydrogen,and Nitrogen were determined in a Perkin-Elmer 240 semi-automaticanalyser.

e. It had an Infra-red analysis of the solid material (1.5mg) dispersedin a standing 16mm diameter potassium chloride disc, having thefollowing absorbances per mg. of solid glycopeptide:

    ______________________________________                                        bond        Wavelength cm.sup.-1                                                                         absorbance/mg                                      ______________________________________                                        CO-NH       1660           0.78                                               C-OH        1050-1070      0.29 *                                             C-H         2850-2950      0.004 *                                            ______________________________________                                         *relative to albumin                                                     

Details of the actual infra-red spectrum obtained are given hereinbelow.

f. Chromatographic fractionation of a hydrolysate of the glycopeptideindicated the presence of certain aminoacids and sugars as follows:

amino acids

ornithine, alanine, valine, leucine and aspartic acid; the hydrolysisbeing effected in 6N HCL in sealed ampoules for 18 hours at 100° C, theresidue being dissolved in water and

Chromatography being effected on Whatman 3 mm paper using ascendingchromatography for 18 to 24 hours with a solvent comprising butanol(60ml), acetic acid (15ml), and water (25ml) followed by developmentwith a ninhydrin spray, for amino acids; and

sugars

galactose, glucose, mannose and trace amounts of arabinose; thehydrolysis being effected in 2N HCl in sealed ampoules for 3 hours at100° C, the residue being dissolved in pyridine and chromatography beingeffected on Whatman 3 mm paper using descending chromatography for 18 to24 hours with a solvent comprising butanol (60ml), pyridine (40ml), andwater (30ml) followed by development with an Aniline-Oxalate spray forsugars (P. Novotny, J. Med. Microbiol. 2 81 (1969));

g. A Biuret test for protein was carried out (Ref. 10) on theglycopeptide which took up a blue colouration which was not washed off.

h. The glycopeptide yielded a positive response to the Anthrone test(Ref. 11), the reaction solution turning blue-green.

EXAMPLE 2 and 3 Isolation of immunostimulating glycopeptide of C. parvumcells

Two further glycopeptide preparations (hereinafter referred to asglycopeptides B and C) were obtained by the process described in Example1, using the same C. parvum strain.

EXAMPLE 4 Isolation of immunostimulating glycopeptide of C. parvum Cells

A further glycopeptide preparation (hereinafter referred to asglycopeptide D) was obtained from the C. parvum strain used in Example 1by the process described in Example 1 but with the following difference:the mixture of lysate, water and phenol was centrifuged at 1,500g for 1hour (instead of at 8,000g for 30 minutes).

EXAMPLE 5 Properties of glycopeptides of Examples 1 and 2

1. Infra-red analyses of glycopeptides A and B dispersed in standard16mm diameter potassium halide discs yielded the following results:

    ______________________________________                                        wave-      Glycopeptide A                                                                              Glycopeptide B                                       length     (1.57mg in KCl disc)                                                                        (1.50mg. in KBr disc)                                cm.sup.-1  A      %Abs    A°                                                                          A    %Abs   A°                          ______________________________________                                        850        min     .12  22         .02   5                                    *1055      max     .68  79    .58  .31  50     .66                            1152       infl.   .37  58         .19  36                                    1200       min     .20  36         .10  20                                    1380       doublet                                                            1405       max     .49  67    .42  .26  45     .55                            1480       min     .26  45         .15  29                                    1545       max     .72  81    .62  .35  55     .74                            1575       min     .64  77         .32  52                                    1605       infl.   .78  83         --   --                                    1660       max     1.17 93    1.00 .47  66     1.00                           1900       base    .03   5         .07  14                                    2920       max     .37  57    .32  .24  42     .51                            3400       max     1.10 92    .94  .50  70     1.06                           ______________________________________                                         *Max. almost flat, 1045-1070 cm.sup.-1                                        min = minimum                                                                 max = maximum                                                                 infl =  inflection                                                            A = absorbance                                                                %Abs = percentage absorption                                                  A° = absorbance relative to peptide band at 1660 cm.sup.-1        

2. Microanalysis of glycopeptide B (under the same conditions as inExample 1. (d)) yielded the following composition: Carbon 39.77%,Hydrogen 6.40%, Nitrogen 8.15%, Ash 2.8%, Silica 0.84% and less than0.2% of each of Phosphorus and Sulphur, the percentage values givenbeing by weight of the dry weight of the glycopeptide.

EXAMPLE 6 Pharmaceutical composition suitable for injection.

The glycopeptide of Example 1 (70mg) was suspended in a solution ofphysiological saline (0.85% w/v aqueous sodium chloride, 10ml)containing 0.01% w/v thiomersalate. The suspension was sterilised by theaddition of 0.1% w/v formalin for 24 hours whereupon the formalin wasremoved by centrifugation at 38,000g for 30 minutes. After washing twicein physiological saline the glycopeptide was then resuspended inphysiological saline (10ml) containing 0.01% w/v thiomersalate anddistributed into 1ml glass ampoules which were then sealed.

EXAMPLE 7 Properties of glycopeptides of Examples 2 & 3

1. Infra-red analyses of glycopeptides B and C dispersed in standard16mm diameter potassium chloride discs yielded the following results.

    __________________________________________________________________________    wave-    Glycopeptide C                                                                            wave-   Glycopeptide D                                   length   (1.45 mg)   length  (1.54mg)                                         cm.sup.-1                                                                              A    %Abs                                                                              A°                                                                        cm.sup.-1                                                                             A    %Abs                                                                              A°                               __________________________________________________________________________    925      .16  31     860  min                                                                              .145 28                                          1063 max .55  72  .44                                                                              1061 max                                                                              .66  78  .66                                     1155 max .33  53     1152 infl                                                                             .36  56.5                                        1202 min .195 36     1200 min                                                                              .19  35                                          1382                                                                               doublet                                                                           .425 62.5                                                                              .34                                                                              1382                                                                              doub                                                                           .48                                                                              67   .48                                         1412 Max .47  66     1410 max                                                                              .47  66                                          1482 min .26  45     1480 min                                                                              .25  44                                          1557 max .86  86  .69                                                                              1570 max                                                                              .74  82  .74                                     1605 min .74  82                                                              1900     .035  8  .33                                                                              1900 base                                                                             .035  8                                          2940 max .41  61     2940 maX                                                                              .38  58  .38                                     3400 max 1.25 94  1.00                                                                             3400 max                                                                              .96  89  .96                                     1660 max 1.25 94  1.00                                                                             1650 max                                                                              1.00 90  1.00                                    __________________________________________________________________________     min = minimum?                                                                max = maximum                                                                 infl = inflection                                                             A = absorbance                                                                %Abs = percentage absorption                                                  A° = absorbance relative to peptide band at 1660 cm.sup.-1        

2. Microanalysis of glycopeptide C (as in Example 1 (d)) yielded thefollowing composition: Carbon 38.51%, Hydrogen 5.81%, Nitrogen 8.22%,Phosphorus 0.23%, Sulphur >0.3%, Ash 4.2% and Silica 1.81%, thepercentage values given being by weight of dry weight of the extract.

EXAMPLE 8 Biological properties of glycopeptide of C.parvum cells

1. Lympho -- recticular stimulating activity

10 Olac mice (Random-bred, W-Swiss females from 16 to 20g bodyweight)each received a single intravenous injection containing the glycopeptideof Example 3 (1.4mg) in 0.85% w/v aqueous sodium chloride (0.2ml). After10 days the mice were sacrificed and their spleens and livers recoveredand weighed. 10 similar control mice each received a single intravenousinjection of aqueous sodium chloride (0.85% w/v) and after 10 days weresacrificed, and their spleens and livers recovered and weighed. Theresults are shown in Table 1 as the means organ weight in mg. correctedfor a 20g mouse body weight.

                  Table 1                                                         ______________________________________                                        Effect of glycopeptide in increasing spleen                                   and liver weights in mice                                                            Spleen Weight (mg)                                                                          Liver Weight (mg)                                        ______________________________________                                        Controls 102             1060                                                 Test Mice                                                                              250             1741                                                 ______________________________________                                    

2. Anti-tumour activity

20 Olac mice (females each of 16-20g bodyweight) were each injectedintraperitoneally with an allogeneic mouse sarcoma 180T/G (10⁵ cells)(Ref.16). 24 hrs later 10 test mice each received a single intravenousinjection containing the glycopeptide of Example 3 (1.4mg) in aqueoussodium chloride (0.85% w/v). The remaining 10 control mice each receiveda single intravenous injection of aqueous sodium chloride (0.85% w/v).

                  Table 2                                                         ______________________________________                                        The effect of glycopeptide extracted from                                     C. parvum on Tumour 180/TG in mice                                            Days from                                                                     glycopeptide Number of mice surviving                                         injection date                                                                             Controls      Test Mice                                          ______________________________________                                        15           10            10                                                 16           10            9                                                  17           9             9                                                  18           6             9                                                  19           4             9                                                  20           4             8                                                  21           3             7                                                  22           2             7                                                  23           2             7                                                  24           2             7                                                  25           2             5                                                  26           1             5                                                  27           1             5                                                  28           1             5                                                  29           1             5                                                  30           1             5                                                  ______________________________________                                    

Mouse fatalities were then recorded over a period of 30 days.

RESULTS

Table 2 shows the effect of the glycopeptide in improving lifeexpectancy of mice injected intraperitoneally with the mouse sarcoma.

EXAMPLE 9 Isolation of Immunostimulating glycopeptide from C.parvumcells

A further glycopeptide preparation (hereinafter referred to asglycopeptide E) was isolated from lysed cells prepared as in Example 1.The isolation procedure of Example 1 was then followed except that thephenolic phase was discarded at the same time as the aqueous phase.Dialysis was effected as before in order to remove residual phenol andyielded a sediment of only two layers: a lower dark coloured layer andan upper white layer. The latter was collected as before to yield theglycopeptide E.

EXAMPLE 10 Isolation of immunostimulating glycopeptide from C.parvumcells

A further glycopeptide preparation (hereinafter referred to asglycopeptide F) was obtained using the C.parvum strain and the processdescribed in Example 1.

EXAMPLE 11 Biological properties of glycopeptides of Example 9 and 10.

The lymphoreticular stimulating activity of glycopeptides E and F wasstudied using the procedure given in Example 8.1, yielding the followingresults:

    ______________________________________                                                       spleen    liver                                                               weight (mg)                                                                             weight (mg)                                          ______________________________________                                        Controls (5)      88         1155                                             Mice receiving 1.0mg                                                          of glycopeptide E (5)                                                                          227         1724                                             Controls (5)      91         1098                                             Mice receiving 1.4mg                                                          of glycopeptide F (10)                                                                         185         1481                                             ______________________________________                                    

The spleen and liver weights given are the mean values. The numbers inbrackets are the numbers of mice involved in the tests.

EXAMPLE 12 Extraction of whole C. parvum cells

C. parvum whole cells were freeze dried and to 20 g dry weight was addeddistilled water (400 ml) and aqueous phenol (90% w/v, 400 ml) both at68° C. The mixture was incubated at 68° C for 15 minutes with constantstirring, cooled in an ice bath and the mixture then centrifuged at 8000g for 30 minutes at 4° C. Four distinct layers were observed in thecentrifuge vessel, a pellet, a lower phenolic phase, a very small amountof a white interphase layer, and an upper aqueous phase. The aqueous andphenolic phases were discarded and the pellet and interphase materialresuspended in a further quantity of distilled water (400 ml) at 68° Ctogether with phenol (400 ml) also at 68° C and re-extracted as before.

Extractions were repeated using fresh water and phenol until the phenollayer was colourless. It was noticeable that the interphase materialincreased in amount with each extraction process.

The final aqueous and phenolic phases were discarded and the pellet andinterphase materials combined and dialysed against running tap water for4 days followed by distilled water at 4° C for a further 3 days in orderto remove the phenol.

After dialysis a sediment of 2 layers was present in the dialysistubing. A lower grey/green oily layer possessing little or noimmunostimulating activity and an upper white layer which possessed theactivity of the present invention.

The upper white layer was collected, after water had been removed fromthe dialysis tubing, to give the glycopeptide. The C. parvum strain wasthe same as in Example 1, namely Wellcome Culture No. C.N. 6134, whichwas obtained from Pasteur Institute Culture No. 4685.

Physical Properties

The glycopeptide possessed the following properties:

a. It was slightly soluble in dimethyl sulphoxide. It was insoluble inwater, phenol, ethanol, methanol, chloroform, dioxan, pyridine,dimethylformamide, diethylene glycol, hexane, acetone, ether andphysiological saline;

b. It was substantially lipid-free (J. Falch et al J. Biol. Chem. 226497 (1957));

c. It had a chemical composition as determined by microanalysis, theamounts representing percentages by weight: Carbon 41.3; Hydrogen 6.4;Nitrogen 10.85; Phosphorus 0; Silica 0; Sulphur below 0.2; and Oxygen,by difference, of the order of 40. Carbon, Hydrogen, and Nitrogen weredetermined in a Perkin-Elmer 240 semi-automatic analyser.

d. It had an Infra-red analysis of the solid material dispersed in astandard 16 mm diameter potassium chloride disc, having absorbances at1055 cm⁻¹ and 1545 and 1660 cm⁻¹ respectively, and an absorbance at 2920cm⁻¹ consistent with the pressure of little or no lipid.

e. Chromatographic fractionation of a hydrolysate of the glycopeptideindicated the presence of certain sugars as follows:

Sugars

galactose, glucose, mannose and trace amounts of arabinose;

the hydrolysis being effected in 2N HCl in sealed ampoules for 3 hoursat 100° C, the residue being dissolved in pyridine and chromatographybeing effected on Whatman 3 mm paper using descending chromatography for18 to 24 hours with a solvent comprising butanol (60 ml), pyridine (40ml), and water (30 ml) followed by development with an Aniline-Oxalatespray for sugars (P. Novotny, J. Med. Microbiol. 2 81 (1969));

f. A Biuret test for protein was carried out (Ref. 10) on theglycopeptide which took up a blue colouration which was not washed off.

g. The glycopeptide yielded a positive response to the Anthrone test(Ref. 11), the reaction solution turning blue-green.

EXAMPLE 13 Biological Properties of glycopeptide of Example 12

A. The lymphoreticular stimulating activity of the glycopeptide wasstudied using the procedure given in Example 8.1, yielding the followingresults:

    ______________________________________                                                       Spleen    Liver                                                               weight mg weight mg                                            ______________________________________                                        Controls          88         1155                                             Test mice        526         2624                                             ______________________________________                                    

The spleen and liver weights given are the mean values.

B. The anti-tumour activity of the glycopeptide was studied.

A test group comprising 10 Olac mice (females each of 16-20 gbodyweight) were each injected intravenously with glycopeptide (0.43 mg)in aqueous sodium chloride (0.85% w/v).

After 7 days both groups of mice were challenged by intraperitonealinjection of an allogeneic mouse sarcoma 180T/G (10¹⁵ cells) Ref. 16.Mouse fatalities were then recorded over a period of 30 days.

RESULTS

Table 3 shows the effect of the glycopeptide in improving lifeexpectancy of mice challenged with the mouse sarcoma.

                  Table 3                                                         ______________________________________                                        The effect of glycopeptide extracted from C. par-                             vum on Tumour 180T/G in mice                                                  Days from   Number of mice surviving                                          Challenge   Controls       Test mice                                          ______________________________________                                        16          9              10                                                 17          8              10                                                 18          7              10                                                 19          7              10                                                 20          7              10                                                 21          5              10                                                 22          2              6                                                  23          1              4                                                  24          1              3                                                  25          1              3                                                  26          1              2                                                  27          1              1                                                  28          1              1                                                  29          1              1                                                  30          1              1                                                  Harmonic mean                                                                 Survival time                                                                 (in days)   21.95          25.6                                               ______________________________________                                    

C. effect of glycopeptide on histamine sensitisation in mice (Ref. 17).

Groups of 10 NIH mice (adult females each of 16-20 g bodyweight) wereinjected with aqueous sodium chloride (0.85% w/v) containing 0; 0.43 or1.4 mg of glycopeptide. After 7 days all groups were challenged withhistamine phosphate and deaths recorded over 24 hours (Ref. 17).

Results

Table 4 shows the effect of the glycopeptide in sensitising the mice tohistamine.

                  Table 4                                                         ______________________________________                                        The effect of glycopeptide extracted from                                     C. parvum in sensitising mice to histamine                                    Dose of     Dose of        number of mice                                     glycopeptide                                                                              histamine      surviving (after                                   (mg per mouse)                                                                            base (mg/mouse)                                                                              24 hours)                                          ______________________________________                                                    0.25           0                                                  1.4         0.125          2                                                              0.0625         7                                                              0.5            1                                                  0.43        0.25           4                                                              0.125          6                                                              10             10                                                 0           5              10                                                 (controls)  1              9                                                              0.5            10                                                 ______________________________________                                    

D. toxicity Data for the Glycopeptide

The following tests of the U.S. Dept. of Health, Education and WelfarePublic Service, Regulations for the Manufacture of Biological ProductsTitle 42 Part 73, Division of Biologies standards, N.I.H. Bethesda, Md.,U.S.A. were carried out on the glycopeptide.

1. Mouse Weight Loss

Groups of 10 male mice (14-16 g) received intraperitoneal injections(0.2 ml volume) of varying dilutions of the whole cells of C. parvum(starting material for obtaining the glycopeptide); the glycopeptideobtained by the phenol and water extraction; and saline. The mice wereweighed daily for 7 days and the weight loss compounded as a percentageof the starting weight.

RESULTS

The mean mouse weights during the test period are given in Table 1 fromwhich it may be seen that:

a. no fatalities were recorded during the test period;

b. that in all cases the starting weights were recovered within therequired 3 days and in fact the starting weights were recovered within 2days of the injection of the test material, in all cases; and

c. no significant differences in the recovery of lost weight were foundbetween the groups injected with the known whole cell C. parvum materialand the groups injected with the new glycopeptide of the presentinvention.

                                      Table 1.                                    __________________________________________________________________________    Bodyweights of mice from mouse weight loss tests for whole                    cell C. parvum and for glycopeptide of the present invention                           Dosage                                                               Material mg per                                                                            Days after injection                                             injected mouse                                                                             0  1  2  3  4  5  6  7                                           __________________________________________________________________________      Saline --  100                                                                              106                                                                              111                                                                              121                                                                              126                                                                              130                                                                              134                                                                              139                                         A.                                                                              C. parvum                                                                            1.4 100                                                                               91                                                                              101                                                                              115                                                                              123                                                                              128                                                                              135                                                                              148                                                  0.47                                                                              100                                                                               99                                                                              107                                                                              119                                                                              124                                                                              126                                                                              131                                                                              134                                           whole cells                                                                          0.16                                                                              100                                                                              103                                                                              106                                                                              119                                                                              125                                                                              129                                                                              135                                                                              139                                           Saline --  100                                                                              106                                                                              116                                                                              126                                                                              135                                                                              141                                                                              149                                                                              156                                         B.                                                                              Glycopeptide                                                                         1.4 100                                                                               90                                                                              106                                                                              111                                                                              125                                                                              131                                                                              137                                                                              142                                                  0.47                                                                              100                                                                               96                                                                              108                                                                              116                                                                              127                                                                              134                                                                              138                                                                              147                                                  0.16                                                                              100                                                                              101                                                                              111                                                                              119                                                                              127                                                                              131                                                                              136                                                                              143                                         __________________________________________________________________________

2. Pyrogenicity

Groups of 2 rabbits received intravenous injections (0.4 ml volume) ofvarying dilutions of whole cells of C.parvum (known material also usedas starting material for obtaining glycopeptide); the glycopeptideobtained by the phenol and water extraction; and saline. Rectaltemperature rise was recorded by thermocouple over 5 hours.

RESULTS

The mean rabbit rectal temperatures recorded over the test period areshown in Table 2, from which it may be seen that the rise in temperaturewas less for those rabbits injected with the glycopeptide than for thoseinjected with the known whole cell C.parvum material, at all the dosagestested.

                  Table 2.                                                        ______________________________________                                        Rectal Temperature from Pyrogenicity tests in rabbits for                     whole cell C. parvum and for glycopeptide of the present                      invention.                                                                    Material    Dose mg     Mean Temperature rise                                 injected    per rabbit  in ° C                                         ______________________________________                                        A.  Saline      --          0.1                                                   C. parvum   2.8         1.55                                                  whole cells 0.93        1.45                                                              0.31        1.2                                               B.  Saline      --          0.2                                                   Glycopeptide                                                                              2.8         1.2                                                               0.93        1.35                                                              0.31        1.0                                               ______________________________________                                    

References

1. Prevot, A.-R., & Fredette, V., (1966) `Manual for the classificationand determination of the anaerobic bacteria`, Lea and Febiger,Philadelphia, U.S.A.

2. prevot, A.-R., J. Retic. Soc. 1 115 (1964)

3. Mackie & McCartney's Handbook of Bacteriology p. 233 Edited by R.Cruickshank, 10th Ed., Livingstone, Edinburgh & London, 1960.

4. ibid p. 192

5. ibid p. 234

6. ibid p. 309

7. ibid p. 308

8. Nature 202 (4930) p. 364-366, 25th April 1966.

9. Adlam C. & M. T. Scott, J. Med. Microbiol. 6 261 (1973).

10. Data for Biochemical Research Ed. Dawson, R.M.C., Daphne C. Elliott,W. H. Elliott & K. M. Jones, 2nd Edn. O.U.P., Oxford 1969 p. 618.

11. Spiro, R. G. Methods in Enzymology 8 3 (1966) (Ed. Neufeld, E. F. &Ginsberg, V., pub. Academic Press New York & London.

12. Adlam, C., Broughton, E. S. & Scott, M. T., Nature, New Biol. 235,219 (1972).

13. Nussenzweig, Ruth S. Expl. Parasitol., 21, 224 (1967).

14. Woodruff, M. F. A. & Boak, J. L., Brit. J. Cancer 20 345 (1966).

15. Sartorelli A. C., Fischer D. S. & Downs W. G., J. Immunology 96 676(1966).

16. Westphal, O. & Jann, K., Methods in Carbohydrate Chemistry 5 83(1966), Ed. Whistler, pub. Academic Press, New York & London.

17. Adlam C., J. Med Microbiol., 6 527 (1973)

What we claim is:
 1. An immunostimulating glycopeptide suitable for usein conferring on mammals or birds a resistance to Sarcoma 180T/G tumourand pathogenic microoganisms which glycopeptide is obtainable from thecell wall of an immunostimulating bacterium belonging to theActinomycetaceae family of Prevot's classification, which glycopeptideis substantially insoluble in water, physiological saline, methanol,ethanol, phenol, chloroform, dioxan, pyridine, dimethylformamide,diethylene glycol, and hexane; has an absorption in the Infra-redspectrum at 1550 and 1660 cm⁻¹ consistent with the presence of peptide;has an absorption in the Infra-red spectrum between 950 and 1100 cm⁻¹with a maximum absorption at 1050 to 1070 cm⁻¹ consistent with thepresence of carbohydrate; has little or no lipid absorption in theInfra-red spectrum at 2850 to 2950 cm⁻¹ ; and which glycopeptide, afterintravenous administration, increases the liver and spleen weight ofmice, and increases the life expectancy of mice with tumours,
 2. Aglycopeptide as claimed in claim 1 wherein the immunostimulatingbacterium is a member of the Corynebacterium genus.
 3. A glycopeptide asclaimed in claim 2 wherein the immunostimulating bacterium is a memberof the Corynebacterium parvum species.
 4. A process for obtaining aglycopeptide as defined in claim 1 comprising the steps of culturing theimmunostimulating Actinomycetaceae bacterial cells; recovering culturedbacterial cells from the culture; and subjecting the cultured bacterialcells to biphasic solvent extraction using one phase comprising waterand a second phase comprising an aryl alcohol to as to yield theglycopeptide as insoluable residue.
 5. A process as claimed in claim 4wherein the cultured bacterial cells are subjected to lysis prior tobiphasic solvent extraction.
 6. A process as claimed in claim 4 whereinthe biphasic solvent extraction comprises the steps of contacting thebacterial cells and/or a lysate thereof with the water and the arylalcohol followed by centrifugation of the resulting mixture, removal ofthe aqueous phase and removal of the alcoholic phase to yield a sedimentcontaining an upper layer comprising a white finely divided suspensionwhich is collected to yield the glycopeptide.
 7. A process as claimed inclaim 6 wherein the aryl alcohol is unsubstituted phenol.
 8. Aglycopeptide as claimed in claim 1 wherein the immunostimulatingbacterium belongs to the strain of the Corynebacterium parvum specieshaving the Wellcome Culture Collection No. CN
 6134. 9. A process asclaimed in claim 4 wherein the immunostimulating bacterial cells are ofthe Corynebacterium genus.
 10. A process as claimed in claim 9 whereinthe immunostimulating bacterial cells are of the Corynebacterium parvumspecies.
 11. A process as claimed in claim 4 wherein theimmunostimulating bacterial cells are of the strain of theCorynebacterium parvum species having the Wellcome Culture CollectionNo. CN 6134.